Our laboratory as well as others has identified an heterogeneous group of intracellular organelles collectively called the endosomal apparatus which participates sequentially in the internalization of insulin, its receptor, as well as other peptide hormones, growth factors and their receptors. It is within endosomes that the highest concentration and total amount of internalized ligand- receptor complexes are found. We hypothesize that within endosomes aspects of hormone action, effected via transmembrane signalling, are initiated and/or maintained. Our past studies documented the in vivo augmentation of insulin receptor kinase activity in endosomes. Of note was our finding that this internalized kinase activity was less ligand dependent in respect to both autophosphorylation and exogenous substrate phosphorylation. We propose to study: (i) the extent of ligand receptor coupling in endosomes; (ii) the regulation of receptor kinase activities at both the cell surface and within the endosomal apparatus; (iii) endogenous substrates for the insulin receptor kinase in endosomes; and (iv) the bioactivity of internalized receptors within endosomes. Thus, morphological and biochemical assessments of ligand-receptor dissociation will be assessed in purified endosomes in order to evaluate the contribution of ligand-receptor coupling to receptor kinase stimulation in the endosome. The kinetics of insulin and EGF receptor phosphorylation in plasma membranes and endosomes will be determined and tryptic fragments of phosphorylated receptors will be resolved and identified by reverse phase HPLC using gas- phase sequencing and region specific antibodies. This will permit evaluation of the roles of receptor autophosphorylation on receptor internalization and activation. As well we shall focus on characterizing endogenous substrates for the insulin receptor- kinase in order to determine any significant association with endosomes. Finally, the ability of endosomes to trigger a bioresponse will be assessed following the microinjection of insulin receptor-enriched endosomes into Xenopus oocytes and EGF receptor-enriched endosomes into NIH 3T3 cells where the progression of oocytes through meiosis and triggering of c myc and c fos gene expression will be evaluated respectively. In this way, we propose to determine the physiological relevance of activated endosomal receptor kinases to peptide hormone action.